RESUMO
One of the main hurdles in the development of new inhaled medicines is the frequent observation of foamy macrophage (FM) responses in non-clinical studies in experimental animals, which raises safety concerns and hinders progress into clinical trials. We have investigated the potential of a novel multi-parameter high content image analysis (HCIA) assay as an in vitro safety screening tool to predict drug induced FM. Rat (NR8383) and human U937-derived alveolar macrophages were exposed in vitro to a panel of model compounds with different biological activity, including inhaled bronchodilators, inhaled corticosteroids (ICS), phospholipidosis inducers and proapoptotic agents. An HCIA was utilized to produce drug-induced cell response profiles based on individual cell health, morphology and lipid content parameters. The profiles of both rat and human macrophage cell lines differentiated between cell responses to marketed inhaled drugs and compounds known to induce phospholipidosis and apoptosis. Hierarchical clustering of the aggregated data allowed identification of distinct cell profiles in response to exposure to phospholipidosis and apoptosis inducers. Additionally, in NR8383 cell responses formed two distinct clusters, associated with increased vacuolation with or without lipid accumulation. U937 cells presented a similar trend but appeared less sensitive to drug exposure and presented a narrower range of responses. These results indicate that our multi-parameter HCIA assay is suitable to generate characteristic drug-induced macrophage response profiles, thus enabling differentiation of foamy macrophage phenotypes associated with phospholipidosis and apoptosis. This approach shows great potential as pre-clinical in vitro screening tool for safety assessment of candidate inhaled medicines.
Assuntos
Macrófagos Alveolares , Macrófagos , Ratos , Humanos , Animais , Macrófagos Alveolares/metabolismo , Células Espumosas , Linhagem Celular , LipídeosRESUMO
Within drug development and pre-clinical trials, a common, significant and poorly understood event is the development of drug-induced lipidosis in tissues and cells. In this manuscript, we describe a mass spectrometry imaging strategy, involving repeated analysis of tissue sections by DESI MS, in positive and negative polarities, using MS and MS/MS modes. We present results of the detected distributions of the administered drug, drug metabolites, lipid molecules and a putative marker of lipidosis, di-docosahexaenoyl (22:6)-bis(monoacylglycerol) phosphate (di-22:6-BMP). A range of strategies have previously been reported for detection, isolation and identification of this compound, which is an isomer of di-docosahexaenoic (22:6 n-3) phosphatidylglycerol (di-22:6 PG), a commonly found lipid that acts as a surfactant in lung tissues. We show that MS imaging using MS/MS can be used to differentiate these compounds of identical mass, based upon the different distributions of abundant fragment ions. Registration of images of these fragments, and detected drugs and metabolites, is presented as a new method for studying drug-induced lipidosis in tissues. Graphical abstract.
Assuntos
Biomarcadores/metabolismo , Lipidoses/induzido quimicamente , Pulmão/diagnóstico por imagem , Espectrometria de Massas/métodos , Amiodarona/efeitos adversos , Animais , Antiarrítmicos/efeitos adversos , Masculino , Ratos Wistar , RoedoresRESUMO
PURPOSE: Progress to the clinic may be delayed or prevented when vacuolated or "foamy" alveolar macrophages are observed during non-clinical inhalation toxicology assessment. The first step in developing methods to study this response in vitro is to characterize macrophage cell lines and their response to drug exposures. METHODS: Human (U937) and rat (NR8383) cell lines and primary rat alveolar macrophages obtained by bronchoalveolar lavage were characterized using high content fluorescence imaging analysis quantification of cell viability, morphometry, and phospholipid and neutral lipid accumulation. RESULTS: Cell health, morphology and lipid content were comparable (p < 0.05) for both cell lines and the primary macrophages in terms of vacuole number, size and lipid content. Responses to amiodarone, a known inducer of phospholipidosis, required analysis of shifts in cell population profiles (the proportion of cells with elevated vacuolation or lipid content) rather than average population data which was insensitive to the changes observed. CONCLUSIONS: A high content image analysis assay was developed and used to provide detailed morphological characterization of rat and human alveolar-like macrophages and their response to a phospholipidosis-inducing agent. This provides a basis for development of assays to predict or understand macrophage vacuolation following inhaled drug exposure.
Assuntos
Amiodarona/farmacologia , Lipídeos/análise , Macrófagos Alveolares/citologia , Macrófagos Alveolares/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Células Espumosas/química , Células Espumosas/citologia , Células Espumosas/efeitos dos fármacos , Células Espumosas/ultraestrutura , Humanos , Macrófagos Alveolares/química , Macrófagos Alveolares/ultraestrutura , Masculino , Imagem Óptica/métodos , Fosfolipídeos/análise , Ratos , Ratos WistarRESUMO
There is a paucity of data describing the impact of salt counterions on the biological performance of inhaled medicines in vivo. The aim of this study was to determine if the coadministration of salt counterions influenced the tissue permeability and airway smooth muscle relaxation potential of salbutamol, formoterol, and salmeterol. The results demonstrated that only salbutamol, when formulated with an excess of the 1-hydroxy-2-naphthoate (1H2NA) counterion, exhibited a superior bronchodilator effect (p < 0.05) compared to salbutamol base. The counterions aspartate, maleate, fumarate, and 1H2NA had no effect on the ability of formoterol or salmeterol to reduce airway resistance in vivo. Studies using guinea pig tracheal sections showed that the salbutamol:1H2NA combination resulted in a significantly faster (p < 0.05) rate of tissue transport compared to salbutamol base. Furthermore, when the relaxant activity of salbutamol was assessed in vitro using electrically stimulated, superfused preparations of guinea pig trachea, the inhibition of contraction by salbutamol in the presence of 1H2NA was greater than with salbutamol base (a total inhibition of 94.13%, p < 0.05). The reason for the modification of salbutamol's behavior upon administration with 1H2NA was assigned to ion-pair formation, which was identified using infrared spectroscopy. Ion-pair formation is known to modify a drug's physicochemical properties, and the data from this study suggested that the choice of counterion in inhaled pharmaceutical salts should be considered carefully as it has the potential to alter drug action in vivo.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Albuterol/química , Albuterol/farmacologia , Naftóis/química , Traqueia/efeitos dos fármacos , Animais , Ácido Aspártico , Cromatografia Líquida de Alta Pressão , Composição de Medicamentos , Fumaratos/metabolismo , Cobaias , Técnicas In Vitro , Masculino , Maleatos/metabolismo , Traqueia/metabolismoRESUMO
'Foamy' alveolar macrophages (FAM) observed in nonclinical toxicology studies during inhaled drug development may indicate drug-induced phospholipidosis, but can also derive from adaptive non-adverse mechanisms. Orally administered amiodarone is currently used as a model of pulmonary phospholipidosis and it was hypothesized that aerosol administration would produce phospholipidosis-induced FAM that could be characterized and used in comparative inhalation toxicology. Han-Wistar rats were given amiodarone via (1) intranasal administration (6.25 mg/kg) on two days, (2) aerosol administration (3 mg/kg) on two days, (3) aerosol administration (10 mg/kg) followed by three days of 30 mg/kg or (4) oral administration (100 mg/kg) for 7 days. Alveolar macrophages in bronchoalveolar lavage were evaluated by differential cell counting and high content fluorescence imaging. Histopathology and mass-spectrometry imaging (MSI) were performed on lung slices. The higher dose aerosolised amiodarone caused transient pulmonary inflammation (p < 0.05), but only oral amiodarone resulted in FAM (p < 0.001). MSI of the lungs of orally treated rats revealed a homogenous distribution of amiodarone and a putative phospholipidosis marker, di-22:6 bis-monoacylglycerol, throughout lung tissue whereas aerosol administration resulted in localization of both compounds around the airway lumen. Thus, unlike oral administration, aerosolised amiodarone failed to produce the expected FAM responses.
RESUMO
Lipid nanocapsules (LNCs) are semi-rigid spherical capsules with a triglyceride core that present a promising formulation option for the pulmonary delivery of drugs with poor aqueous solubility. Whilst the biodistribution of LNCs of different size has been studied following intravenous administration, the fate of LNCs following pulmonary delivery has not been reported. We investigated quantitatively whether lung inflammation affects the clearance of 50nm lipid nanocapsules, or is exacerbated by their pulmonary administration. Studies were conducted in mice with lipopolysaccharide-induced lung inflammation compared to healthy controls. Particle deposition and nanocapsule clearance kinetics were measured by single photon emission computed tomography/computed tomography (SPECT/CT) imaging over 48 h. A significantly lower lung dose of (111)In-LNC50 was achieved in the lipopolysaccharide (LPS)-treated animals compared with healthy controls (p<0.001). When normalised to the delivered lung dose, the clearance kinetics of (111)In-LNC50 from the lungs fit a first order model with an elimination half-life of 10.5±0.9h (R(2)=0.995) and 10.6±0.3h (R(2)=1.000) for healthy and inflamed lungs respectively (n=3). In contrast, (111)In-diethylene triamine pentaacetic acid (DTPA), a small hydrophilic molecule, was cleared rapidly from the lungs with the majority of the dose absorbed within 20min of administration. Biodistribution to lungs, stomach-intestine, liver, trachea-throat and blood at the end of the imaging period was unaltered by lung inflammation. This study demonstrated that lung clearance and whole body distribution of lipid nanocapsules were unaffected by the presence of acute lung inflammation.
Assuntos
Lipídeos/administração & dosagem , Lipídeos/farmacocinética , Pulmão/metabolismo , Nanocápsulas/administração & dosagem , Pneumonia/metabolismo , Administração por Inalação , Animais , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Meia-Vida , Cinética , Lipopolissacarídeos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Camundongos Endogâmicos BALB C , Pneumonia/induzido quimicamente , Pneumonia/patologia , Distribuição TecidualRESUMO
To date, the role of nanoparticle surface hydrophobicity has not been investigated quantitatively in relation to pulmonary biocompatibility. A panel of nanoparticles spanning three different biomaterial types, pegylated lipid nanocapsules, polyvinyl acetate (PVAc) and polystyrene nanoparticles, were characterized for size, surface charge, and stability in biofluids. Surface hydrophobicity of five nanoparticles (50-150nm) was quantified using hydrophobic interaction chromatography (HIC) and classified using a purpose-developed hydrophobicity scale: the HIC index, range from 0.00 (hydrophilic) to 1.00 (hydrophobic). This enabled the relationship between the nanomaterial HIC index value and acute lung inflammation after pulmonary administration to mice to be investigated. The nanomaterials with low HIC index values (between 0.50 and 0.64) elicited little or no inflammation at low (22cm(2)) or high (220cm(2)) nanoparticle surface area doses per animal, whereas equivalent surface area doses of the two nanoparticles with high HIC index values (0.88-0.96) induced neutrophil infiltration, elevation of pro-inflammatory cytokines and adverse histopathology findings. In summary, a HIC index is reported that provides a versatile, discriminatory, and widely available measure of nanoparticle surface hydrophobicity. The avoidance of high (HIC index>~0.8) surface hydrophobicity appears to be important for the design of safe nanomedicines for inhalation therapy.